Over the last several years, our group has engineered microbial opsins, naturally occurring light-activated ion channels and pumps, for use in neuroscience, where they are in widespread use for assessing the causal contribution of a given cell type or pathway to neural circuit functions, behaviors, and pathologies. We have distributed these tools to perhaps thousands of labs, with often hundreds of requests before each publication appears. In this competing renewal R01 grant, we propose to extend this optogenetic toolbox in fundamentally new ways, in order to meet the challenges posed by outstanding sets of neuroscience questions. First (Aim 1), we propose to develop a toolbox of optogenetic tools optimized for developmental neuroscience, including optogenetic silencers that are red-shifted enough (i.e., even into the infrared) and light-sensitive enough that they can support noninvasive optogenetic silencing (important because of the difficulty of using optical fibers in the size-changing developing mammalian brain), and opsins that can express extremely quickly (important because many opsins can take days to weeks to express, too long to be used to study development in many mammalian scenarios). These molecules will also have broad impact outside of developmental neuroscience: for example, the ability to noninvasively silence neurons will be useful for mammalian studies where brain lesion, aseptic compromise, glial activation, or inflammation from optical fiber insertion is undesired (e.g., in many disease studies), and in chronic experiments not compatible with long-term probe insertion. Second (Aim 2), in order to optogenetically mimic endogenous ion channels and thus enable the causal testing of how specific ion conductances contribute to neural computation, we will use a novel automated microscopy methodology that we have developed, to perform a high-throughput screen for mutants that have more selective ion permeability, aiming to make light-driven potassium and sodium channels, which can mimic endogenous conductances in neurons. These molecules will enable the study, for example, of how specific conductances at specific points on neural dendrites or axons, contribute to the conversion of neural input into neural output. Third (Aim 3), in order to understand how more complex neural activity patterns contribute to brain functions, we will enable optogenetics to be used to assess how multiple (e.g., >2) sets of neurons work together in a neural circuit, by improving the multiplexing capability of optogenetics to enable three different neuron classes to be independently controlled by three different colors of light. We will also enable clean two- photon activation of single cells embedded within intact brain networks, by restricting optogenetic protein expression just to cell bodies, so that attempts to stimulate single cells in densely labeled brain tissue do not artifactually stimulate nearby axons or dendrites passing by, or forming synapses upon cell bodies of interest. Finally (Aim 4), we will implement implantable waveguides equipped with active focusing and beam-steering, so that optogenetic control of neurons deep in the mammalian brain, with single cell resolution, is possible.